Glucocorticoids act by interacting with the glucocorticoid receptor (GR) in the cytoplasm, which then translocates to the nucleus and binds the GR responsive elements (GRE) in the promoter regions of target genes. Glucocorticoid induced leucine zipper (GILZ) (SEQ ID NO: 1) is a glucocorticoid-inducible gene with six GRE elements. Overexpression of GILZ suppresses activated T cells by inhibiting transactivation of nuclear factor kappa B (NF-κB), the master regulator of inflammatory responses. The anti-inflammatory activity of synthetic glucocorticoids mediated by preventing NF-κB transactivation has been attributed to the induced upregulation of GILZ (SEQ ID NO: 1).
Multiple sclerosis (MS) is a common chronic inflammatory and demyelinating disease of the central nervous system. MS affects young and middle aged adults, and women in particular, imposing significant economic costs on individuals and the community in terms of loss of productivity and efficiency. MS is widely recognized as a complex disease driven by dysregulated immunity accompanied by relapsing and remitting clinical manifestations. There are many symptoms associated with MS, including weakness, fatigue, sensory loss, paresthesias, optic neuritis, diplopia, ataxia, vertigo, pain, lhermitte, demenital, visual loss, and inflammation. The pivotal role played by NF-κB in the pathogenesis of MS is well established. Peptides derived from the NF-κB essential modulator (NEMO), the NEMO binding unit (NBD) of IKKβ proteins and the phosphorylation sites of p65 have been evaluated to directly target the NF-κB multisubunit complex.
The ability of currently available therapeutic agents to modify the disease course in MS is modest at best. Hence, there is a need for developing more effective disease-modifying therapies. MS is widely recognized as a complex disease driven by dysregulated immunity accompanied by relapsing and remitting clinical manifestations. Since the antigenic trigger of MS is not definitively identified, most disease modifying agents are designed to modulate the inflammatory response in the periphery and in the brain. Therapies under development include strategies that deplete lymphocytes and those that mediate immunomodulation. Since depletion could potentially cause global immunosuppression, strategies of immunomodulation are preferable.
The profound anti-inflammatory activity of synthetic glucocorticoids, combined with their ability to induce lymphocyte apoptosis place them among the most commonly prescribed drugs in the management of relapse in MS. However, poor efficacy in reducing the frequency or severity of relapse and the complications of serious side-effects compromise the continuous or long-term use of steroids. Hence, treatments that harness the therapeutic effects of glucocorticoids with a better benefit-to-risk ratio than traditional steroids are needed.
GILZ, originally identified as a glucocorticoid-inducible gene, has been shown to interact with the p65 subunit of NF-κB in activated T cells. Since activated p65 is present only in stimulated cells, the GILZ mimetics may mediate selective inhibition of activated cells without causing widespread immunosuppression. Preliminary results suggest that treatment with exogenous GILZ (SEQ ID NO: 1) suppresses antigen activated T cell responses in vitro. Low molecular weight GILZ mimetics that bind the p65 protein with optimal kinetics are characterized. Advantages of peptides such as GILZ mimetics as therapeutics include non-immunogenicity with potential for long-term use, greater permeability to cross tissue barriers, and cost-effectiveness.
One of the actions of glucocorticoids is to modulate the transcription of multiple genes involved in immune response. The anti-inflammatory effect is largely attributed to the inhibition of nuclear factor kappa B (NF-κB), a regulator of inflammatory responses. GILZ having an amino acid sequence MNTEMYQTPMEVAVYQLHNFSISFFSSLLGGDVVSVKLDNSASGASVVAIDNKIEQAM DLVKNHLMYAVREEVEILKEQIRELVEKNSQLERENTLLKTLASPEQLEKFQSCLSPEEP AP ESPQVPEAPGGSAV (SEQ ID NO: 1), is a glucocorticoid induced gene that binds the p65 subunit of NF-κB to inhibit its nuclear translocation.
NF-κB is a heterodimer of p50 and p65 that remains as an inactive complex with inhibitory proteins such as IκB in the cytoplasm of resting T cells. Following T cell activation, p65 is released from the inhibitory complex, translocates to the nucleus and mediates transactivation of inflammatory genes. Gene profiling studies revealed the presence of elevated p65 in the peripheral blood monocytes and in the pathologic lesions in MS. It is believed herein that therapeutic agents that sequester activated p65 within the cytoplasm will suppress transactivation of inflammatory cytokines and ameliorate disease in MS. Based on analysis of mutational and functional studies, it is believed herein that the proline rich carboxy terminus of GILZ (SEQ ID NO: 1) interacts physically with the p65 subunit of NF-κB. Proline rich regions (PRR) are often localized in the solvent exposed regions of proteins involved in transient interactions such as signaling or cytoskeletal rearrangements. Hence PRRs provide target sites for developing inhibitors of transient protein-protein interactions. Rationally designed peptide mimetics of the proline rich p65 binding interface of GILZ (SEQ ID NO: 1) may sequester p65 within the cytoplasm in activated T cells and suppress inflammation related to MS. Without being bound by any particular theory, such peptide mimetics may act as a glucocorticoid mimetic and/or as an NF-κB inhibitor.
Knowledge derived from the primary structure of GILZ (SEQ ID NO: 1) and its interaction with the p65 subunit of NF-κB is applied to the design of novel peptide agents, the GILZ mimetics. The carboxy terminus of GILZ (GILZ-COOH) consists of 35 residues (residues 100-134 of GILZ); LASPEQLEKFQSCLSPEEPAPESPQVPEAPGGSAV (SEQ ID NO: 2). Significantly, it has three “PXXP” motifs: PEEP (SEQ ID NO: 3), PESP (SEQ ID NO: 4), and PEAP (SEQ ID NO: 5). The structural and functional significance of this motif in transient intermolecular interactions such as cytoskeletal rearrangement and signaling is well established. Secondary structure prediction of GILZ-COOH by the nearest neighboring neural network suggested a helical conformation between 103P-S111 indicating a region with significant propensity to bind DNA. Hence, synthetic peptides corresponding to one or more proline rich regions of GILZ may be effective GILZ mimetics.
Localized in the solvent exposed domains, the role of PRRs is to bring proteins together so as to make subsequent interactions more probable. This is particularly true for interactions between functionally important proline and conserved hydrophobic residues in the interface of its binding partner. Notably, the transactivation domain of p65 that potentially interacts with the GILZ protein presents two highly conserved phenylalanine residues; F534 and F542 which together with the conserved acidic residues at Asp531 and Asp533 and phosphorylation sites at Ser529 and Ser536 constitute the critical residues for p65 transactivation. Data from these observations are integrated with that from residue interface propensity to introduce rational amino acid substitutions and/or truncations in the GILZ-WT so as to design GILZ mimetics with optimal p65 binding efficacy. Additional parameters considered to increase the drug like properties of GILZ mimetics include solvent mediated contact potential, accessible surface volume/residue, log interface residue propensity, weighted hydrophobicity and solvation potential.
Conventionally, the proof of therapeutic manipulation of specific intermolecular interactions is derived from studies using fusion proteins and/or monoclonal antibodies of the interacting molecules such as Abatacept (CTLA4 IgG1Fc), Alefacept (LFA-3-IgG1Fc), Denileukin diftitox (recombinant IL-2) and Etanercept (TNFR-IgG1Fc) or adalimumab/Certolizumab pegol/infliximab (anti-TNF-α mAb), Natalizumab (anti-α4 integrin,) and Efalizumab (anti-CD11a mAb) respectively. These agents target interactions occurring at cell surface and/or in the extracellular environment. In contrast, the GILZ:p65 interaction occurs in the cytoplasm necessitating intracellular delivery of potential modulating agent(s). In this context, low molecular weight peptides that are permeable and better amenable for intracellular delivery than large proteins represent attractive alternatives.
Without being bound by any particular theory, it is believed that because activated p65 is present only in activated T cells, intracellular delivery of GILZ mimetics will suppress pro-inflammatory responses by sequestering activated p65 and facilitate skewing towards anti-inflammatory responses. An advantage of lower molecular weight peptides as therapeutic agents include increased permeability for intracellular delivery compared to larger proteins.
The following numbered embodiments are contemplated and are non-limiting:
1. A pharmaceutical composition comprising a polypeptide from about 6 to about 35 amino acid residues, the polypeptide comprising 1 to 3 tetrapeptides having the sequence of PXXP, wherein                P is proline; and        X is any amino acid.        
2. The pharmaceutical composition of clause 1, wherein at least one tetrapeptide comprises the amino acid sequence of SEQ ID NO: 3.
3. The pharmaceutical composition of clause 1 or clause 2, wherein at least one tetrapeptide comprises the amino acid sequence of SEQ ID NO: 4.
4. The pharmaceutical composition of any one of clauses 1 to 3, wherein at least one tetrapeptide comprises the amino acid sequence of SEQ ID NO: 5.
5. The pharmaceutical composition of clause 1, wherein the polypeptide comprises the amino acid sequence of CLSPEEPAPESPQVPEAPGGSAV (SEQ ID NO: 6).
6. The pharmaceutical composition of any one of clauses 1 to 5, wherein the polypeptide comprises a polyproline helical conformation.
7. The pharmaceutical composition of any one of clauses 1 to 6 further comprising a cell penetrating peptide.
8. The pharmaceutical composition of clause 7, wherein the cell penetrating peptide is selected from the group consisting of Penetratin, Pep-1, Pep-2, VP22, pVEC, pISL, hCT derived peptide, LL-37, Mouse PrP, Transportan, TP10, Arg11, MAP, MPG, KALA, ppTG1, and ppTG20.
9. The pharmaceutical composition of clause 7, wherein the cell penetrating peptide is a bipartite peptide (K16ApoE) consisting of a polylysine segment linked with the apolipoprotein-E peptide.
10. The pharmaceutical composition of clause 7, wherein the cell penetrating peptide is Pep-1.
11. The pharmaceutical composition of any one of clauses 1 to 10, wherein the composition suppresses p65 binding.
12. The pharmaceutical composition of any one of clauses 1 to 11, wherein the composition suppresses p65 activation.
13. The pharmaceutical composition of any one of clauses 1 to 12, wherein the composition inhibits NF-κB translocation to the nucleus of a cell.
14. The pharmaceutical composition of any one of clauses 1 to 13, wherein the composition suppresses T cell response.
15. The pharmaceutical composition of any one of clauses 1 to 14, wherein the composition suppresses T-bet transcription.
16. The pharmaceutical composition of any one of clauses 1 to 15, wherein the composition suppresses a pro-inflammatory cytokine.
17. The pharmaceutical composition of any one of clauses 1 to 16, wherein the composition suppresses a cytokine associated with Th-1 response.
18. The pharmaceutical composition of clause 17, wherein the cytokine is selected from the group consisting of IL-12, IL-17, IFN-γ, TNF-α, and IL-23.
19. The pharmaceutical composition of clause 17, wherein the cytokine is IL-12.
20. The pharmaceutical composition of clause 17, wherein the cytokine is IL-17.
21. The pharmaceutical composition of clause 17, wherein the cytokine is IFN-γ.
22. The pharmaceutical composition of clause 17, wherein the cytokine is TNF-α.
23. The pharmaceutical composition of clause 17, wherein the cytokine is IL-23.
24. The pharmaceutical composition of any one of clauses 1 to 23, wherein the composition enhances a cytokine associated with Th-2 response.
25. The pharmaceutical composition of clause 24, wherein the cytokine is selected from the group consisting of IL-4, IL-10 and TGF-β.
26. The pharmaceutical composition of clause 24, wherein the cytokine is IL-4.
27. The pharmaceutical composition of clause 24, wherein the cytokine is IL-10.
28. The pharmaceutical composition of clause 24, wherein the cytokine is TGF-β.
29. The pharmaceutical composition of any one of clauses 1 to 28, wherein the composition upregulates a Th-2 specific transcriptional factor.
30. The pharmaceutical composition of clause 29, wherein the Th-2 specific transcriptional factor is STAT-6.
31. The pharmaceutical composition of clause 29, wherein the Th-2 specific transcriptional factor is GATA-3.
32. The pharmaceutical composition of any one of clauses 1 to 31, wherein the composition is associated with a Th-2 bias in the Th-1/Th-2 balance.
33. The pharmaceutical composition of any one of clauses 1 to 32, for use in the treatment of multiple sclerosis.
34. The pharmaceutical composition of any one of clauses 1 to 33, wherein the composition reduces a symptom associated with multiple sclerosis.
35. The pharmaceutical composition of clause 34, wherein the symptom associated with multiple sclerosis is an inflammatory symptom.
36. A pharmaceutical composition comprising a polypeptide from about 6 to about 35 amino acid residues, the polypeptide comprising 1 to 3 tetrapeptides having the sequence of PEXP, wherein                P is proline;        E is glutamic acid; and        X is any amino acid.        
37. The pharmaceutical composition of clause 36, wherein at least one tetrapeptide comprises the amino acid sequence of SEQ ID NO: 3.
38. The pharmaceutical composition of clause 36 or clause 37, wherein at least one tetrapeptide comprises the amino acid sequence of SEQ ID NO: 4.
39. The pharmaceutical composition of any one of clauses 36 to 38, wherein at least one tetrapeptide comprises the amino acid sequence of SEQ ID NO: 5.
40. The pharmaceutical composition of clause 36, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 6.
41. The pharmaceutical composition of any one of clauses 36 to 40, wherein the polypeptide comprises a polyproline helical conformation.
42. The pharmaceutical composition of any one of clauses 36 to 41 further comprising a cell penetrating peptide.
43. The pharmaceutical composition of clause 42, wherein the cell penetrating peptide is selected from the group consisting of Penetratin, Pep-1, Pep-2, VP22, pVEC, pISL, hCT derived peptide, LL-37, Mouse PrP, Transportan, TP10, Arg11, MAP, MPG, KALA, ppTG1, and ppTG20.
44. The pharmaceutical composition of clause 42, wherein the cell penetrating peptide is a bipartite peptide (K16ApoE) consisting of a polylysine segment linked with the apolipoprotein-E peptide.
45. The pharmaceutical composition of clause 42, wherein the cell penetrating peptide is Pep-1.
46. The pharmaceutical composition of any one of clauses 36 to 45, wherein the composition suppresses p65 binding.
47. The pharmaceutical composition of any one of clauses 36 to 46, wherein the composition suppresses p65 activation.
48. The pharmaceutical composition of any one of clauses 36 to 47, wherein the composition inhibits NF-κB translocation to the nucleus of a cell.
49. The pharmaceutical composition of any one of clauses 36 to 48, wherein the composition suppresses T cell response.
50. The pharmaceutical composition of any one of clauses 36 to 49, wherein the composition suppresses T-bet transcription.
51. The pharmaceutical composition of any one of clauses 36 to 50, wherein the composition suppresses a pro-inflammatory cytokine.
52. The pharmaceutical composition of any one of clauses 36 to 51, wherein the composition suppresses a cytokine associated with Th-1 response.
53. The pharmaceutical composition of clause 52, wherein the cytokine is selected from the group consisting of IL-12, IL-17, IFN-γ, TNF-α, and IL-23.
54. The pharmaceutical composition of clause 52, wherein the cytokine is IL-12.
55. The pharmaceutical composition of clause 52, wherein the cytokine is IL-17.
56. The pharmaceutical composition of clause 52, wherein the cytokine is IFN-γ.
57. The pharmaceutical composition of clause 52, wherein the cytokine is TNF-α.
58. The pharmaceutical composition of clause 52, wherein the cytokine is IL-23.
59. The pharmaceutical composition of any one of clauses 36 to 48, wherein the composition enhances a cytokine associated with Th-2 response.
60. The pharmaceutical composition of clause 59, wherein the cytokine is selected from the group consisting of IL-4, IL-10 and TGF-β.
61. The pharmaceutical composition of clause 59, wherein the cytokine is IL-4.
62. The pharmaceutical composition of clause 59, wherein the cytokine is IL-10.
63. The pharmaceutical composition of clause 59, wherein the cytokine is TGF-β.
64. The pharmaceutical composition of any one of clauses 36 to 63, wherein the composition upregulates a Th-2 specific transcriptional factor.
65. The pharmaceutical composition of clause 64, wherein the Th-2 specific transcriptional factor is STAT-6.
66. The pharmaceutical composition of clause 64, wherein the Th-2 specific transcriptional factor is GATA-3.
67. The pharmaceutical composition of any one of clauses 36 to 66, wherein the composition is associated with a Th-2 bias in the Th-1/Th-2 balance.
68. The pharmaceutical composition of any one of clauses 36 to 67, for use in the treatment of multiple sclerosis.
69. The pharmaceutical composition of any one of clauses 36 to 68, wherein the composition reduces a symptom associated with multiple sclerosis.
70. The pharmaceutical composition of clause 69, wherein the symptom associated with multiple sclerosis is an inflammatory symptom.
71. A method of treating multiple sclerosis, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of clauses 1 to 70.
72. The method of clause 71, wherein the method suppresses p65 binding in the patient.
73. The method of clause 71 or clause 72, wherein the method suppresses p65 activation in the patient.
74. The method of any one of clauses 71 to 73, wherein the method inhibits NF-κB translocation to the nucleus of a cell in the patient.
75. The method of any one of clauses 71 to 74, wherein the method suppresses T cell response in the patient.
76. The method of any one of clauses 71 to 75, wherein the method suppresses T-bet transcription in the patient.
77. The method of any one of clauses 71 to 76, wherein the method suppresses a pro-inflammatory cytokine in the patient.
78. The method of any one of clauses 71 to 77, wherein the method suppresses a cytokine associated with Th-1 response in the patient.
79. The method of clause 78, wherein the cytokine is selected from the group consisting of IL-12, IL-17, IFN-γ, TNF-α, and IL-23.
80. The method of clause 78, wherein the cytokine is IL-12.
81. The method of clause 78, wherein the cytokine is IL-17.
82. The method of clause 78, wherein the cytokine is IFN-γ.
83. The method of clause 78, wherein the cytokine is TNF-α.
84. The method of clause 78, wherein the cytokine is IL-23.
85. The method of any one of clauses 71 to 84, wherein the method enhances a cytokine associated with Th-2 response in the patient.
86. The method of clause 85, wherein the cytokine is selected from the group consisting of IL-4, IL-10 and TGF-β.
87. The method of clause 85, wherein the cytokine is IL-4.
88. The method of clause 85, wherein the cytokine is IL-10.
89. The method of clause 85, wherein the cytokine is TGF-β.
90. The method of any one of clauses 71 to 89, wherein the method upregulates a Th-2 specific transcriptional factor in the patient.
91. The method of clause 89, wherein the Th-2 specific transcriptional factor is STAT-6.
92. The method of clause 89, wherein the Th-2 specific transcriptional factor is GATA-3.
93. The method of any one of clauses 71 to 92, wherein the method is associated with a Th-2 bias in the Th-1/Th-2 balance.
94. The method of any one of clauses 71 to 93, wherein the administration is an injection.
95. The method of clause 94, wherein the injection is selected from the group consisting of intraarticular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous injections.
96. The method of clause 94, wherein the injection is an intravenous injection.
97. The method of any one of clauses 71 to 96, wherein the administration is performed as a single dose administration.
98. The method of any one of clauses 71 to 96, wherein the administration is performed as a multiple dose administration.
99. A pharmaceutical formulation comprising the pharmaceutical composition of any one of clauses 1 to 70.
100. The pharmaceutical formulation of clause 99 further comprising a pharmaceutically acceptable carrier.
101. The pharmaceutical formulation of clause 99 or clause 100 optionally including one or more other therapeutic ingredients.
102. The pharmaceutical formulation of any one of clauses 99 to 101 wherein the formulation is a single unit dose.
103. A lyophilisate or powder of the pharmaceutical formulation of any one of clauses 99 to 102.
104. An aqueous solution produced by dissolving the lyophilisate or powder of clause 103 in water.